Development of a zebrafish embryo teratogenicity assay and quantitative prediction model

The Dechorinated Zebrafish Embryo Developmental toxicity assay was originally developed from a training set of 31 compounds and reported to be 87% concordant with in vivo teratogenicity data (Brannen, K. C., Panzica-Kelly, J. M., Danberry, T. L., and Augustine-Rauch, K. A. (2010). Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. Birth Defects Res. 89, 66-77.). The assay includes scoring larva treated in a concentration range for malformations of specific morphological structures/organ systems. The model includes identifying a no-adverse-effect-level (NOAEL) and the concentration resulting in 25% lethality (LC25) at 5 days postfertilization. An LC25/NOAEL ratio ≥10 classifies a compound positive for teratogenic potential. A consortium effort evaluated a modified version of this assay which involved enzymatic chorion treatment instead of manual dissection and used experimental replicates for final classification. The modified assay achieved an 85% overall predictivity (Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I. Reprod. Toxicol. 33, 155-164.). The objective of this study was to perform a thorough performance evaluation of the dechorinated assay by repeating the original training set and testing additional compounds in experimental replicates. When the initial training set was repeated with inclusion of experimental replicates, the overall predictivity was 83%. Model performance was tested with an additional 34 compounds and achieved overall predictivity of 74%. When the training and test sets were combined (63 compounds) the assay's final sensitivity was 83% and the specificity was 71%. Total predictivity was 78% with relatively balanced predictivity for nonteratogens (77%) and teratogens (78%). The chorion-off assay achieved superior sensitivity (83%) compared with sensitivity (63-74%) reported by consortium efforts testing a similar assay with chorion-intact embryos (Ball, J. S., Stedman, D. B., Hillegass, J. M., Zhang, C. X., Panzica-Kelly, J., Coburn, A., Enright, B. P., Tornesi, B., Amouzadeh, H. R., Hetheridge, M., et al. (2014). Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay. Toxicol. Sci. 139, 210-219.). Additional protocol modifications were made to increase assay throughput.

The transcriptomic Point of Departure (tPOD) is increasingly used in ecotoxicology to derive quantitative endpoints from RNA sequencing studies. Utilizing transcriptomic data in zebrafish embryos as a New Approach Methodology (NAM) is beneficial due to its acknowledgment as an alternative to animal testing under EU Directive 2010/63/EU. Transcriptomic profiles are available in zebrafish for various modes of action (MoA). The limited literature available suggest that tPOD values from Fish Embryo Toxicity (FET) tests align with, but are generally lower than, No Observed Effect Concentrations (NOEC) from long-term chronic fish toxicity tests.In studies with the androgenic hormone androstenedione in a Fish Sexual Development Test (FSDT), a significant shift in the sex ratio towards males was noted at all test concentrations, making it impossible to determine a NOEC (NOEC <4.34 μg/L). To avoid additional animal testing in a repetition of the FSDT and adhere to the 3Rs principle (replacement, reduction, and refinement), a modified zebrafish FET (zFET) was conducted aiming to determine a regulatory acceptable effect threshold. This involved lower concentration ranges (20 to 6105 ng/L), overlapping with the masculinization-observed concentrations in the FSDT. The tPOD analysis in zFET showed consistent results with previous FSDT findings, observing strong expression changes in androgen-dependent genes at higher concentrations but not at lower ones, demonstrating a concentration-response relationship.The tPOD values for androstenedione were determined as 24 ng/L (10th percentile), 60 ng/L (20th gene), and 69 ng/L (1st peak). The 10th percentile tPOD value in zFET was 200 times lower than the lowest concentration in the FSDT. Comparing the tPOD values to literature suggests their potential to inform on the NOEC range in FSDT tests.

Stereoisomeric developmental toxicity of orysastrobin and its isomeric metabolite in zebrafish (Danio rerio) embryos: Cardiac abnormal development by orysastrobin.

Valproic acid (VPA) is a branched-chain carboxylic acid with broad anticonvulsant activity. Its spectrum of activity is thought to be mediated by combined molecular effects on intercellular Na+ currents, neuron K+ channels, and inhibition of gamma-aminobutyric acid (GABA) transaminase (1,2). VPA is metabolized in the liver by cytochrome P-450 oxidase. Hepatotoxicity related to valproate therapy has been reported in certain individuals and appears to be caused by the accumulation of toxic metabolites, which may include 4-en-valproate (3). The hepatotoxicity has been ascribed to an inherited or acquired deficiency in the cytochrome P-450 enzyme-dependent beta-oxidation pathway, and is inducible by other drugs such as phenobarbital or phenytoin (4). Fatal hepatic failure can result, with a disproportionately large number of such cases observed in patients less than two years old with neurologic abnormalities, severe seizures, and multiple anti-epileptic drug therapy (3,5,6,7). However, neonatal hepatic toxicity in humans coincident with intrauterine VPA exposure has rarely been reported (8,9,10); these cases were never reversible, andconfounding etiologic variables could not be excluded. In pregnancy, VPA readily crosses the placenta and accumulates with fetal blood concentrations greater than those in the mother (11). It is a known human teratogen primarily associated with neural tube developmental defects such as spina bifida. A specific "fetal valproate syndrome," marked by fetal growth deficiency, developmental delay, and an increased incidence of craniofacial, cardiovascular, and digital abnormalities, has also been reported (1,3,12,13,14). Furthermore, a dose-dependent relationship in the occurrence of major malformations and minor anomalies has been suggested (15). Major malformations reported include persistent patent ductus arteriosus, trigonocephaly, aplasia of the first ribs, dysplasia of the sternum, meningomyelocele, aplasia of the radius, and congenital hip dislocation. Minor malformations included brachycephaly, dysmorphic facial features, long thin fingers, and inverted or accessory nipples. The teratogenic potential is thought to be mediated by a secondary zinc deficiency induced by complexing with VPA (16), as well as by inhibition of microsomal epoxide hydrolase (17), which may enhance the teratogenic potential of concomitant anticonvulsant therapy. Numerous animal models have been developed for the study of VPA. VPA has been shown to have equal teratogenic potential as trimethadione in mice (18). Skeletal abnormalities, delayed parturition, and postnatal growth (5) have been reported in both mice and rats receiving VPA. In addition, Paulson and Paulson (19) noted defects of the palate, eyes, heart, and limb buds in animal models. Animal models for a VPA-associated cholestasis, however, are lacking.

6-Mercaptopurine riboside (6-MPr) is used as a cytostatic chemotherapeutic. The teratogenic potential in rodents has been well known for several decades. In this study, the teratogenic risk of low doses of 6-MPr in NMRI mice was estimated based on a dose-response study. The effective doses corresponding to the incidences of 5%, 1%, and 0.1% were calculated using the probit and Weibull model. The evaluation was performed on the basis of both the fetus and the litter by evaluating the variable all gross structural abnormalities. From these experiments, benchmark doses were obtained which were used in low-dose risk assessment to define a reference dose. Depending on the biometrical model and the statistical unit used, values between 1.9 and 5.2 mg/kg (benchmark ED1) and 3.8 and 6.7 mg/kg (benchmark ED5) were obtained. These values were compared to the no observed adverse effect level (NOAEL) which was determined experimentally. The NOAEL was found to be 5 mg/kg, which is quite similar to the ED5 benchmark doses.

Modification and quantification of in vivo EROD live-imaging with zebrafish (Danio rerio) embryos to detect both induction and inhibition of CYP1A

CRISPR-Cas9 technology is routinely applied for targeted mutagenesis in model organisms and cell lines. Recent studies indicate that the prokaryotic CRISPR-Cas9 system is affected by eukaryotic chromatin structures. Here, we show that the likelihood of successful mutagenesis correlates with transcript levels during early development in zebrafish (Danio rerio) embryos. In an experimental setting, we found that guide RNAs differ in their onset of mutagenesis activity in vivo. Furthermore, some guide RNAs with high in vitro activity possessed poor mutagenesis activity in vivo, suggesting the presence of factors that limit the mutagenesis in vivo. Using open access datasets generated from early developmental stages of the zebrafish, and guide RNAs selected from the CRISPRz database, we provide further evidence for an association between gene expression during early development and the success of CRISPR-Cas9 mutagenesis in zebrafish embryos. In order to further inspect the effect of chromatin on CRISPR-Cas9 mutagenesis, we analysed the relationship of selected chromatin features on CRISPR-Cas9 mutagenesis efficiency using publicly available data from zebrafish embryos. We found a correlation between chromatin openness and the efficiency of CRISPR-Cas9 mutagenesis. These results indicate that CRISPR-Cas9 mutagenesis is influenced by chromatin accessibility in zebrafish embryos.

Ecotoxicological assessment of cimetidine and determination of its potential for endocrine disruption using three test organisms: Daphnia magna, Moina macrocopa, and Danio rerio

Variations of enzymatic activity and gene expression in zebrafish (Danio rerio) embryos co-exposed to zearalenone and fumonisin B1

Taxifolin mitigates oxidative DNA damage in vitro and protects zebrafish (Danio rerio) embryos against cadmium toxicity

Acute and developmental toxic effects of mono-halogenated and halomethyl naphthalenes on zebrafish (Danio rerio) embryos: Cardiac malformation after 2-bromomethyl naphthalene exposure

Pectin is a polymer commonly found in the fruit peels. Pectin-mediated gold nanoparticles (AuNPs) were fabricated and characterized by spectroscopic and microscopic analysis. The transmission electron microscopy revealed the presence of polymorphic and polydisperse nanoparticles in the size range of 4–18 nm. Cytotoxicity studies using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide on Gambusia affinis skin cell line demonstrated that there is no significant toxicity at lower concentrations. Zebrafish (Danio rerio) embryos were used to study the effect and distribution of AuNPs in vivo and the toxicological parameters (mortality, heart beats per min and hatching rate) were recorded. A concentration-dependent rise in mortality was also observed in AuNPs-treated embryos. Furthermore, the acridine orange staining was used to assess the genotoxicity of pectin-mediated AuNPs on the zebrafish embryos. The results indicated that these particles did not express toxicity to the zebrafish embryonic development at lower concentrations.

Thiram, an oxidized dimer of dithiocarbamate, has fungicidal and ectoparasiticidal roles. This study aimed to determine the effects of thiram on the development of zebrafish (ZF) embryos. The developmental toxicity test was performed in accordance with the OECD 236 test guidelines, and ZF embryos were subjected to several thiram concentrations and a DMSO (0.01%) control. Subsequently, embryo mortalities and developmental anomalies were evaluated at different hours post fertilization (hpf). Thiram was highly toxic to ZF, with calculated median lethal concentrations (LC50) of thiram at 48 and 96 h as 13.10 ± 2.17 and 8.87 ± 2.09 μg/L, respectively. Thiram-treated embryos/larvae exhibited a variety of deformities, such as abnormal somites, reduced eye pigment, abnormal tail shape, yolk sac edema, hatching defects, and curved spines, with a median effective concentration (EC50) of 3.88 ± 1.23, 5.04 ± 1.82, 6.23 ± 0.92, 5.24 ± 2.22, 1.39 ± 0.25, and 2.60 ± 0.82 μg/L, respectively. Teratogenic index (TI) values ranged from 1.42 to 6.66 for the scored deformities. At 48 hpf, the average heartbeat of the control group was 177.20 ± 5.63 per minute, while the highest thiram-treated group (40 μg/L) was 99.50 ± 18.12 per minute. In addition, cardiac-related issues, such as pericardial edema and abnormal blood flow, were observed in thiram-treated ZF embryos. Overall, these findings suggest that thiram is teratogenic to ZF.

BCR-ABL, a fusion protein kinase, is a druggable target exclusively expressed in patients with chronic myeloid leukemia (CML). Several anti-leukemia medicines targeting this protein have been developed in recent years. However, therapeutic options are limited for CML patients bearing multiple BCR-ABL1 mutations. Ponatinib (PON), a potent tyrosinase inhibitor, was one of the approved drugs for managing BCR-ABL1 T315I mutant disease. However, treatment of patients with PON reported severe side effects related to cardiovascular events. Asciminib (ASC) was the first allosteric inhibitor approved to target the myristoyl pocket of BCR-ABL protein to inhibit protein activity. The different mechanism of inhibition opens the possibility of co-exposure with both medicines. Reports on cardiovascular side effects due to the combination use of PON + ASC in pre-clinical and clinical studies are minimal. Thus, this study aimed to observe the potential cardiovascular-related side effect after co-exposure to ASC and PON using zebrafish as an animal model. In this study, zebrafish were acutely exposed to both compounds. The cardiovascular physiology parameters and gene expression related to cardiovascular development were evaluated. We demonstrate that combining ASC with PON at no observed effect concentration (NOEC) did not cause any significant change in the cardiac performance parameter in zebrafish. However, a significant increase in nkx2.5 expression level and a substantial decrease in blood flow velocity were recorded, suggesting that combining these compounds at NOEC can cause mild cardiovascular-related side effects.

Deltamethrin (DM) is a broad-spectrum insecticide that is widely used to control agricultural pests. Recently, DM has posed a potential threat to the health of infants and young children, and this is because of the environmental and food pollution that is caused by the extensive use of DM. In this study, zebrafish (Danio rerio) embryos were used as experimental animals to quantify the behavioral changes of larvae induced by DM and explore the relationship between DM and acetylcholinesterase activity. The results showed that DM caused an increase in coiling movement, heart rate, and apoptosis in the brain in early zebrafish embryos or larvae. It also caused a decrease in the expression of acetylcholinesterase-associated genes and the activity of acetylcholinesterase, which also led to an increase in the acetylcholinesterase content. The transcriptome data also showed that low concentration DM induced acetylcholine-related gene signaling pathways. The above results suggest that low doses of DM may induce neurodegeneration because DM exposure inhibits acetylcholinesterase, leading to brain cell apoptosis and behavioral changes in the zebrafish embryos or larvae. Micro-injection of zebrafish embryos at the 2-4 cell stage knocked down or overexpressed the acetylcholinesterase gene showed that the behavior and enzyme activity of zebrafish had some effects. This study explored the relationship between acetylcholinesterase and the change in zebrafish behavior caused by DM exposure to provide a basis for the treatment of DM poisoning in the aquaculture.